Concentrations Of Levamisole Research Articles

The activation of nicotinic acetylcholine receptors in the nematode parasite Ascaris suum by the application of levamisole to the cytoplasmic surface of muscle membrane

AbstractThe patch‐clamp technique was used to examine activation by levamisole of nicotinic acetylcholine receptors in muscle vesicle preparations from Ascaris suum. Previous experiments have shown that levamisole, when applied at concentrations greater than 30 μM to the extracellular surface of the membrane, activated channels which showed characteristics of a voltage‐sensitive open channel‐block and desensitisation. In this study initial experiments were performed where channels were activated with levamisole (2 μM) applied in the patch‐pipette. Isolated inside‐out patches were used. In addition, levamisole (30‐926 μM) was added to the cytoplasmic membrane surface via the bath solution. In eight out of nine experiments, addition of levamisole to the bath solution resulted in an increase in channel activity, a voltage‐sensitive open channel‐block and desensitisation. The open channel‐block occurred at hyperpolarised potentials, an observation consistent with levamisole (a cationic substance) blocking the channel from the extracellular surface. Thus it was concluded that levamisole crossed from the cytoplasmic side of the membrane, via the lipid phase, to the extracellular surface of the patch. In the presence of high cytoplasmic concentrations of levamisole, open channel‐block was not observed at depolarised potentials, suggesting channel asymmetry.

Levamisole‐activated single‐channel currents from muscle of the nematode parasite Ascaris suum

1. The patch-clamp technique was used to examine levamisole-activated channels in muscle vesicles from Ascaris suum. Cell-attached and isolated inside-out patches were used. 2. Levamisole (1-90 microM), applied to the extracellular surface, activated channels which had apparent mean open-times in the range 0.80-2.85 ms and linear I/V relationships with conductances in the range 19-46 pS. Ion-replacement experiments showed the channels to be cation selective. 3. The kinetics of the channels were analysed. Generally open- and closed-time distributions were best fitted by two, and three exponentials respectively, indicating the presence of at least two open states and at least three closed states. The distributions of burst-times were best-fitted by two exponentials. 4. Channel open- and burst-times were voltage-sensitive: at low levamisole concentrations (1-10 microM), they increased with hyperpolarization. At higher concentrations of levamisole (30 microM and 90 microM) flickering channel-block was observed at hyperpolarized potentials. Using a simple channel-block model, values for the blocking dissociation constant, KB were determined as 123 microM at -50 mV, 46 microM at -75 mV and 9.4 microM at -100 mV. 5. At the higher concentration of levamisole (30 microM and 90 microM) long closed-times separating 'clusters' of bursts were observed, at both hyperpolarized and depolarized membrane potentials and this was interpreted as desensitization.

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.

Immunological Effects of Levamisole In Vitro

Levamisole, an anthelminthic drug with immunological properties, has recently been reported to have antitumor activity when administered with 5-fluorouracil in patients with Duke's C colorectal carcinoma. The mechanism of this antitumor effect is unknown, but has been postulated to be related to levamisole's immunomodulatory properties. To define further the immunomodulatory activities of levamisole, we studied the in vitro effects of levamisole on monocyte and lymphocyte cytotoxicity, activation, and proliferation; induction of cytokine-induced proteins; and expression of tumor-associated antigens. Experiments utilized peripheral blood mononuclear cells from normal donors incubated in the presence of increasing concentrations of levamisole (0.1 to 100 micrograms/ml). Levamisole had no consistent effect on induction of 2',5'-oligoadenylate synthetase activity or indoleamine-2,3-dioxygenase activity, or production of tumor necrosis factor. Levamisole had no effect on monocyte cytotoxicity or expression of HLA-DR, HLA-DQ, HLA-DP, and the Fc receptor. Similarly, levamisole had no significant effect on NK or LAK cytotoxicity or the immunological activation of T-lymphocytes, assessed by expression of CD3, CD4, CD8, CD16, CD25, and CD56. Proliferation of lymphocytes from normal donors, patients with benign polyps, and patients with malignancies, with or without IL-2 or irradiated LS174T cells, was not significantly increased overall. No significant enhancement in the expression of three tumor-associated antigens (880364, NRCO-4, and ING-1) and the intercellular adhesion molecule-1 (ICAM-1) antigen on four human cancer cell lines was observed following in vitro exposure to levamisole. We conclude that levamisole is not a potent modulator of the immune parameters we examined, and that the mechanism behind the unique clinical interaction between levamisole and 5-fluorouracil in colorectal carcinoma remains to be identified.

Levamisole in the Adjuvant Treatment of Colon Cancer

The chemistry, pharmacology, pharmacokinetics, assay methodologies, adverse effects, and dosage of levamisole are described, and the clinical studies of levamisole therapy in patients with colorectal carcinoma are reviewed. Levamisole is a synthetic, orally active agent that has antihelmintic and immunomodulatory properties. It is capable of inducing T-cell differentiation and restoring depressed effector functions of peripheral lymphocytes and phagocytes to normal. The drug is well absorbed from the gastrointestinal tract after oral administration and is extensively metabolized by the liver. Gas chromatography and high-performance liquid chromatography are the most common methods used to measure concentrations of levamisole in biologic fluids. Levamisole combined with fluorouracil has been associated with a one-third reduction in recurrence and risk of death in patients with surgically resected Dukes stage C colon cancer; this combination is now recommended as standard therapy in these patients. Uses in patients with rectal carcinoma, Dukes stage B colon cancer, metastatic colon cancer, other malignancies, or nonmalignant disorders remain investigational. Common adverse effects include nausea, abdominal pain, vomiting, diarrhea, metallic or altered taste, flulike symptoms, mood elevation, insomnia, hyperalertness, dizziness, and headache. The most serious adverse effect associated with levamisole is granulocytopenia. The FDA-approved dosage of levamisole is 50 mg orally every eight hours for three days every two weeks. Levamisole therapy is to be initiated no earlier than 7 and no later than 30 days after surgery and is to be continued for one year. Levamisole combined with fluorouracil has been associated with a one-third reduction in recurrence and risk of death in patients with resected stage C colon cancer. Further research is needed to more clearly define the mechanism of action, optimum dose and scheduling, and clinical efficacy of levamisole in treating other malignancies.

Toxicity and tissue residue depletion of levamisole hydrochloride in young goats

SUMMARY Toxicity and tissue residue depletion studies were conducted in young goats, using an oral drench formulation of levamisole hydrochloride. In the target animal toxicity study, 3 groups of 5 goats each were given levamisole orally to provide approximately 11.88, 23.76, or 35.64 mg of levamisole HCl/kg/d for 3 consecutive days; a fourth group of 5 goats served as untreated controls. Blood samples were taken for analysis of levamisole 1 day prior to dosing and 1, 2, 3, 4, and 7 days following the third dose. At the 35.64-mg/kg dose, 2 of 5 goats responded with typical cholinergic signs of toxicosis on each of the 3 days of dosing. The times for the onset of clinical signs of toxicosis ranged from 18 to 63 minutes, with an average duration of 32 minutes. Administration of 23.76 mg of levamisole HCl/kg resulted in hyperactive behavior in 1 of 5 goats only on the first day of dosing; no abnormal behavior was observed in any of the 5 goats following the second or third dose of levamisole HCl at 23.76 mg/kg. Untoward effects were not seen in the 5 goats dosed at 11.88 mg of levamisole HCl/kg or in the controls during the 3-day dosing period or in the following 7-day observation period. Overall, the observed signs of toxicosis did not become more severe, affect more goats, or persist for a longer period on subsequent dosing days. In the 35.64-mg/kg/d group of the toxicity study, parent drug was detected in the serum of all 4 goats for which assaying was done 1 day after dosing and in the serum of 1 of the 4 goats for which assay was done 2 days after dosing. Only 1 of the 3 goats given 23.67 mg of levamisole HCl/kg/d had a detectable serum concentration of levamisole 1 day after dosing, and none of the goats had detectable serum levamisole concentrations 2 days after the final dose was given. One of the 3 goats for which assaying followed dosing of 11.88 mg/kg/d had measurable levamisole serum concentrations that persisted for 4 days after dosing; the apparent serum half-life of elimination for levamisole in this goat was 19.4 hours. Serum concentrations of levamisole at 1 day after dosing did not correlate with signs of toxicosis in goats immediately following dosing. Liver, kidney, and muscle samples from 25 goats given a single oral dose of 11.88 mg levamisole HCl/kg were analyzed for drug residues 1, 2, 3, 4, and 7 days after dosing (5 goats/sampling time). Within goats, the highest concentrations of levamisole were found in the liver, followed by kidney and then muscle. Depletion of levamisole from the liver was modeled as a single exponential decay function; using means, or an estimate of the 95th percentile of the population, from days 1, 2, 3, and 4 after dosing resulted in values for apparent half-lives of elimination of 1.4 and 1.2 days, respectively. Adjusting the means and 95th percentile estimates by eliminating observations > 3 sd from the mean had little effect on the apparent half-lives of elimination (1.3 and 1.6 days), although the degree of fit was improved 17 and 28%, respectively. Fitting of the outlier values of levamisole liver concentrations to a similar monoexponential equation resulted in an estimate of the apparent half-life of elimination of 2.4 days. Levamisole concentrations in kidney and muscle decreased too rapidly to model their elimination rates. On the basis of serum kinetics of levamisole in 1 goat (of 10) for which the serum was assayed and liver depletion rates of levamisole estimated from 8 (of 25) goats, we concluded that goats may possess a genetic polymorphism with respect to levamisole metabolism and could be considered either normal or slow eliminators. Regardless of levamisole phenotype, however, liver residues of levamisole were estimated to be < 100 μg/kg for 9 days following a single, oral dose of 11.88 mg of levamisole HCl/kg.

Reliability and reproducibility of the larval paralysis test as an in vitro method for the detection of anthelmintic resistance of nematodes against levamisole and morantel tartrate

In order to study the reliability of the larval paralysis test as an in vitro for the detection of resistance of nematodes to levamisole and morantel tartrate, the influence of different parameters was evaluated using resistant and susceptible Ostertagia ostertagi strains. The operator, the sample (10% of the larvae present in the suspension), the incubation time (24, 48 or 72 h), the incubation temperature (20 or 25°C) and the observation period of the larvae (5 or 15 s) had no statistically significant influence on the test results. Statistical differences were obtained only when L 3 larvae of different ages (1 or 2 months) were used. Reversibility of the paralysis did not occur when concentrations of levamisole of ⩽200 μg ml −1 or morantel tartrate of <2000 μg ml −1 were used. The reproducibility of the test was fairly good, with a mean standard deviation of 21.3% for the LC 50 values. Morantel resistance in a strain of O. ostertagi was not confirmed as such by the larval paralysis test. The resistance factor was ⩽1, in spite of an efficacy of morantel of 77.4% as shown in vivo.

Effect of levamisole on chemotaxis of granulocytes from uremic patients.

Uremic granulocyte chemotaxis was assessed in the presence of four different concentrations of levamisole. Chemotactic responsiveness of uremic granulocytes was significantly decreased compared to normal, both in the absence of levamisole and with all levamisole concentrations tested. However, with 10(-3) and 10(-4) M levamisole concentrations, uremic granulocyte chemotaxis was similar to that of normal granulocytes without levamisole. Defective chemotactic activity of granulocytes may play a role in the increased susceptibility of uremic patients to infections. Pharmacological correction of this defect may improve the patients' ability to cope with infections.

Potentiation of interleukin-2 activity by levamisole and imidazole

Levamisole, a known antihelminthic drug, was able to affect IL-2 dependent proliferation of several T lymphocyte clones. Although levamisole did not replace IL-2 and had no effect at saturating IL-2 concentrations, it reduced by 2- to 4-fold the dose of IL-2 required to give 50% of the maximal DNA synthesis, the optimal concentration of levamisole being between 1 to 3 × 10 −4 M. This effect of levamisole was not due to an alteration of the kinetics of thymidine incorporation. Imidazole and other imidazole ring containing compounds, but not thiol-containing compounds, had similar effects to those of levamisole, suggesting a role of the imidazole ring in the potentiation of IL-2 activity.

Determination of levamisole hydrochloride with HgI 2−4 by a turbidimetric method and flow-injection analysis

This paper is concerned with the use of ion-association compounds in the analysis of pharmaceutical samples by FIA. The usual extraction into an organic phase is avoided by using turbidimetric detection. Determination of levamisole with HgI 2− 4 has been developed as a practical example: the experimental variables were optimized by the modified simplex method. The calibration graph is linear over the levamisole concentration range 7–32 μ g ml . The reproducibility (rsd) and injection sample rate are 0.9% and 80/hr, respectively.

Novel assay and pharmacokinetics of levamisole and p-hydroxylevamisole in human plasma and urine.

A new gas chromatographic method was developed for the quantification of levamisole in human plasma and urine, using a nitrogen-phosphorus flame ionization detector. The adsorption of the drug onto glass was prevented by treating the glassware with a siliconizing agent. The sensitivity of the assay was 10 ng ml-1 and as low as 2 ng ml-1 can be detected in plasma. The urinary metabolite p-hydroxylevamisole was analysed by high performance liquid chromatography with ultraviolet detection. The sensitivity of this assay was 0.50 micrograms ml-1. Plasma and urinary concentrations of levamisole were determined in 10 healthy volunteers including seven men and three women following the administration of a single 150 mg dose of levamisole. Levamisole was rapidly absorbed (tmax 1.5 h), giving a peak plasma concentration of 716.7 +/- 217.5 ng ml-1. The plasma elimination half-life of levamisole was 5.6 +/- 2.5 h. Only 3.2 +/- 2.9 per cent of the oral dose was recovered as unchanged drug in the urine, suggesting the importance of clearance of levamisole by routes other than the kidney, and most probably by hepatic metabolism. The urinary concentrations of p-hydroxylevamisole were determined before and after hydrolysis of the urine samples with beta-glucuronidase, and the level of conjugation of the metabolite with glucuronic acid was then estimated. Cumulative recovery of the metabolite accounted for 1.6 +/- 1.1 per cent and 12.4 +/- 5.5 per cent of the oral dose of levamisole before and after hydrolysis, respectively, indicating that p-hydroxylation is a relatively important route of metabolism of levamisole, and that the p-hydroxylated metabolite is excreted mainly in conjugation with glucuronic acid. Except for the absorption rate of levamisole which is approximately twice as rapid in women as in men, there is no marked difference in the pharmacokinetics of levamisole between healthy men and women.

Effect of Levamisole on the Response of Broilers to Coccidiosis Vaccination

The effect of levamisole on the response of commercial broilers to coccidiosis vaccination was assessed. The concentration, frequency, and timing of levamisole were varied in three separate experiments to determine optimum conditions for potentiating the response of broilers to a single oral dose of commercial coccidiosis vaccine at 7 days of age. In all instances, levamisole was found to improve development of immunity to coccidiosis. Birds that received levamisole by intraperitoneal (IP) injection had better weight gains for 3 weeks following coccidiosis vaccination and improved resistance to challenge with Eimeria tenella when compared with vaccinated chickens that did not receive levamisole. However, levamisole given at .25 mg/kg body weight by IP injection 3 days prior to the coccidiosis vaccine resulted in the greatest potentiation of the immune response. Performance parameters following challenge included weight gain, oocyst production, fecal droppings, and livability. Findings confirm and extend observations regarding the ability of levamisole to potentiate vaccination responses in chickens.

Comparison of three routes of administration of a water soluble anthelmintic: levamisole

The plasma concentration of levamisole was determined by liquid chromatography in three groups of 20 heifers treated with levamisole by intramuscular administration, drenching and in drinking water respectively. Maximum concentrations were observed at three hours (1.10 +/- 0.16 microgram/ml) and six hours (0.64 +/- 0.06 microgram/ml) after intramuscular administration and drenching respectively. Large individual variations in plasma concentrations were observed following treatment in drinking water with four animals showing maximum concentrations below 0.1 microgram/ml. Variation in water intake therefore makes the latter method less reliable.

Pharmacokinetics of levamisole in healthy subjects and cancer patients.

Eleven healthy volunteers (six men, five women) and twelve cancer patients (six men, six women) were given single oral doses (2.5 mg/kg body weight) of levamisole, an immunostimulant agent. In a second experiment eleven healthy et volunteers (six men, five women) and twelve cancer patients (six men, six women) were given single oral doses (5 mg/kg body weight) of levamisole. The levamisole concentrations in plasma (24 h blood sampling) were measured by gas chromatography after extraction. A mono-compartment open model was proposed, and some parameters were calculated. The main results from these two experiments are: the drug was absorbed well, the peak levels of levamisole in the plasma were reached after 1.0 – 2.0 hrs and ranged between 0,8 (μg and 1.6 μg/ml with the 2.5 mg and 5 mg/kg body weight doses respectively (concentrations requiredin vitro to produce an immunological effect); the plasma half-life of levamisole calculated from the decline in the concentration curves varied between 3.3 h and 5.1 h; the absorbed fractions were 0.7 and clearance 310 ml/min; twenty-four hours after medication, the levamisole concentrations in plasma were only 6 % of the peak levels.

Lysosomal enzymes and metabolic activity of polymorphonuclear leukocytes from patients with systemic lupus erythematosus and from experimental animals after levamisole treatment

The activity of beta-D-glucuronidase (BDG) was lowered and the activity of myeloperoxidase (MPO) was elevated in polymorphonuclear leukocytes (PMNs) of SLE patients compared with normal subjects. After levamisole treatment, the activities of BDG, MPO, and lysozyme rose in PMNs of SLE patients. A higher activity of lysozyme was also observed in peritoneal PMNs of rabbits after levamisole administration. During incubation of human phagocytizing and non-phagocytizing PMNs, no effects of levamisole at concentrations of 10(-3) to 10(-5) M were observed on the release of lysosomal enzymes. These concentrations of levamisole also did not influence INT reductase activity and production of superoxide by normal human PMNs in the presence of zymosan particles. These findings suggest that levamisole might have an effect on the actual level of lysosomal enzymes in PMNs rather than on their release.

The effect of levamisole on peripheral T-lymphocytes of patients with malignant lymphomas.

The effect of various concentrations (0.015 - 10 micrograms/ml) of Levamisole (LMS) on the peripheral lymphocytes of patients with malignant lymphomas (ML) and normal donors was investigated in vitro. The parameters studied include: E rosettes forming cells (total T lymphocytes), active E rosettes (early T lymphocytes) and DNA synthesis induced by pitogens PHA and Con A. LMS improved significantly lymphocyte response both in patients with ML and normal donors when the cells were stimulated by Con A. In both groups no significant effect was observed on the response to PHA nor on the percentage of E-rosettes, whereas the mean number of active E rosettes was significantly increased on all concentrations of the drug. While in the normal subjects a positive statistical correlation between active E-rosettes and Con A response was observed, in patients with ML an inverse correlation was found. This latter correlation was partially reversed by LMS. These results indicate that LMS, both in normals and in patients with ML, interacts with a specific cell subset (early rosetting lymphocytes), with different functions in normal and pathological situations. The drug shows an effect on the immunological handicap of lymphocytes of patients with ML.

Effect of levamisole on pokeweed mitogen stimulation of immunoglobulin production in vitro.

The effects of levamisole (LMS) on immunoglobulin (Ig) production were studied in vitro using peripheral blood lymphocytes from normal subjects stimulated with pokeweed mitogen (PWM). Cells were cultured for 9 days with varying concentrations of LMS and PWM, and immunoglobulin secretion in the supernatants was quantified by solid phase radioimmunoassay. The results showed that 1) the effect of LMS in vitro depends upon the degree of lymphocyte stimulation by PWM. When PWM stimulation is optimal, typical pharmacologic concentrations of LMS (0.5 micro/ml) decrease both IgM and IgG production by 50%. However, at lower suboptimal doses of PWM, LMS, at similar concentrations, enhances immunoglobulin production by 24% (p less than 0.01). Unstimulated lymphocytes are not affected by LMS. 2) The target cell upon which LMS acts is present among a T subpopulation that lacks the Fc receptor for aggregated rabbit IgG (T gamma -negative). We suggest that the diverse effects of LMS on autoimmune disease in vivo may depend upon both the size and degree of activation of the T gamma -negative lymphocyte pool.

Levamisole potentiation of antigen specific lymphocyte blastogenic response in [formula omitted][formula omitted] exposed but nonresponsive cattle

Incubation of Brucella abortus (field strain) infected and strain 19 vaccinated bovine peripheral blood lymphocytes with B. abortus antigen and levamisole caused a consistently significant increase in [ 3H] thymidine uptake when compared to cultures without levamisole. Levamisole did not potentiate B. abortus -induced blastogenic response of lymphocytes from non-exposed cattle. A dose response study showed that 10 μg/culture induced maximum potentiation of B. abortus -induced lymphocyte stimulation. Using the 10 μg/well concentration of levamisole, further studies were conducted to determine the net potentiation of the blastogenic responses in lymphocytes from B. abortus (field strain) infected cattle, B. abortus strain 19 vaccinated but nonresponsive and non-exposed cattle. Levamisole significantly potentiated the B. abortus -induced lymphocyte blastogenesis in lymphocytes from unresponsive cattle.

Levamisole stimulation of neutrophil chemotaxis and chemokinesis by protection of both the leucoattractant and the cellular chemotactic response from inactivation by the peroxidase/hydrogen peroxide/halide system in vitro.

The effects of levamisole, at concentrations known to stimulate neutrophil motility, on neutrophil post-phagocytic metabolic activity were investigated. Levamisole at these concentrations caused inhibition of hexose monophosphate shunt (HMS) activity, superoxide production, hydrogen peroxide generation and myeloperoxidase (MPO), mediated iodination of ingested Candida albicans,. The inhibition of MPO-mediated iodination was not solely due to lack of H2O availability as a result of decreased HMS activity but also to a primary inhibition of iodination as shown in a cell-free system with horse-radish peroxidase (HRP) and added H2O2 and sodium iodide. Further experiments were designed to investigate possible relationships between stimulation and motility and levamisole-induced inhibition of superoxide generation and peroxidase-mediated iodination. These showed that the peroxidase/halide/H2O2 system caused inactivation of both the leucoattractant and neutrophil chemotactic responsiveness. However, concentrations of levamisole, which stimulate motility and inhibit superoxide production and peroxidase-mediated iodination, protected both the leucoattractant response and the ability of the cell to respond to the leucoattractant from inactivation by the HRP/H2O2/iodide system.

Effect of levamisole on lysosomal enzyme release from polymorphonuclear leukocytes and intracellular levels of cAMP and cGMP after phagocytosis of monosodium urate crystals.

In order to study the influence of levamisole on lysosomal enzyme release, human polymorphonuclear (PMN) leukocytes were incubated with 0.5 mg/ml monosodium urate crystals. The release of neutral proteinase, beta glucuronidase, myeloperoxidase and lactic dehydrogenase as cytoplasmatic marker was measured. Preincubation of PMN with various concentrations of levamisole lasting 1 h resulted in a decrease of all enzymes released. Intracellular levels of both cyclic nucleotides--cAMP and cGMP--rose in PMN preincubated with levamisole, in comparison with untreated cells.